Most bacterial cells are surrounded with a cell wall matrix made of peptidoglycan (PG). This exoskeletal layer fortifies the cell membrane against internal osmotic pressure and is essential for cell integrity. Because it is the target for many widely used antibiotics like penicillin and vancomycin, understanding the biogenesis of PG continues to be of great practical significance. With the rising incidence of antibiotic resistant infections in the United States and abroad, it is now more important than ever to discover new vulnerabilities in essential pathways like PG assembly so that they can be exploited for the development of next generation antibacterial therapies. In this regard, most studies of PG biogenesis over the years have focused on the activities of PG synthase enzymes called penicillin-binding proteins (PBPs), the targets of penicillin. In recent years, however, it has become clear that enzymes capable of cleaving bonds in the PG network are equally important for proper PG biogenesis. These so-called PG hydrolases are potentially dangerous enzymes. If left unchecked, they can damage the cell wall and induce cell lysis. Thus, the regulatory mechanisms controlling PG hydrolases represent attractive targets for new classes of lysis-inducing antibiotics. To better understand these controls, we focused much of the previous funding period on identifying regulators governing PG hydrolases with an emphasis on cell wall amidases required for cell division in gram-negative bacteria. Using Escherichia coli as our model organism, we discovered that the amidase enzymes are all autoinhibited by a regulatory domain that occludes their active site. To promote cell division, we found that these factors require activation by division proteins with LytM domains called EnvC and NlpD. We further showed that amidase activation by EnvC is controlled by the ABC-transporter like complex FtsEX, suggesting the exciting possibility that FtsEX may be a conformational regulator that uses the regulatory power of nucleotide binding and hydrolysis to control PG cleavage at the cell surface. Although these discoveries represent great progress in our understanding of PG hydrolase regulation, the precise mechanisms of amidase activation by the LytM factors and the control of PG hydrolase activity by FtsEX remain unclear. It is also not known how the PG remodeling events catalyzed by these systems are properly coordinated with other major activities of the division apparatus. Uncovering these mechanisms will therefore be a major goal of the next funding period. We will also leverage our expertise in studies of amidase regulation to investigate the function and regulation of PG hydrolases involved in other important aspects of PG biogenesis. The proposed experiments will build on the strong foundation laid in the initial funding period and allow us to continue revealing novel biological mechanisms relevant to future antibiotic development.